Hybridization method to detect a cDNA encoding a human phospholemman-like protein (HPLP)

ABSTRACT

The present invention provides a novel human phospholemman-like protein (HPLP) and the polynucleotides which identify and encode HPLP. The invention provides for genetically engineered expression vectors and host cells comprising the nucleic acid sequence encoding HPLP and for a method for producing the protein. The invention also provides pharmaceutical compositions containing HPLP and the use of such compositions for the prevention or treatment of diseases associated with the expression of HPLP. Additionally, the invention provides antisense molecules to HPLP and their use in the treatment of diseases associated with the expression of HPLP. The invention also provides diagnostic assays which utilize polynucleotides which hybridize with naturally occurring sequences encoding HPLP and antibodies which specifically bind to the protein.

This application is a divisional application of U.S. application Ser.No. 08/725,531 filed Oct. 3, 1996, now U.S. Pat. No. 5,756,310.

FIELD OF THE INVENTION

The present invention relates to nucleic acid and amino acid sequencesof a novel human phospholemman-like protein and to the use of thesesequences in the diagnosis, study, prevention, and treatment of disease.

BACKGROUND OF THE INVENTION

Phospholemman (PLM) is the major plasmalemmal substrate forcAMP-dependent protein kinase (cAMPK) and protein kinase C (PKC). Canineand murine PLM are expressed at high levels in heart, skeletal muscle,and liver, and at low levels in breast, brain, lung, stomach, kidney,and colon (Palmer C et al (1991) J Biol Chem 266: 11126-11130; Moorman JR et al (1992) J Biol Chem 267:14551-14554). PLM is a membrane proteinwhich consists of 72 amino acids and has a calculated molecular weightof 8409. The native protein has an apparent molecular weight of 15 kdalfrom polyacrylamide gel electrophoresis. A distinguishing feature of PLMis its highly basic nature, with a calculated isoelectric point of 9.7(Palmer C et al, supra). PLM consists of an acidic extracellularamino-terminal domain, a single uncharged transmembrane domain, and anextremely basic cytoplasmic carboxy-terminal domain. The cytoplasmicdomain contains consensus cAMPK and PKC phosphorylation sites. Thephosphorylation of PLM by PKC and cAMPK is regulated by insulin andadrenaline, respectively (Walaas S et al (1994) Biochem J 304:635-640).PLM phosphorylation in cardiac muscle occurs after activation of eitherα- or β-adrenergic receptors, and correlates with an increase incontractility (Lindemann J P (1986) J Biol Chem 261:4860-4867).

Expression of PLM in Xenopus oocytes injected with PLM mRNA coincideswith the appearance of voltage-activated chloride currents (Moorman etal, supra). Immunoaffinity-purified recombinant PLM added to planarphospholipid bilayers produces unitary anion currents (Moorman J Ret al(1995) Nature 377:737-740). The high selectivity of the PLM channel forthe sulfonic amino acid taurine suggests that PLM channels link signaltransduction cascades to cell volume regulation. The investigatorsreport that PLM is the smallest membrane protein known to form an ionchannel (Moorman et al (1995), supra).

Mat-8, an 8-kDa transmembrane protein related to PLM, is expressed inmurine breast tumor lines transformed by Neu or Ras oncoproteins.Morrison B W et al ((1994) Oncogene 9:3417-3426) proposed that Mat-8 isa marker of the cell type preferentially transformed by neu or v-Ha-rasoncogenes. A human Mat-8 homolog is expressed both in primary breasttumors and in breast tumor cell lines. Murine Mat-8 is also expressed inuterus, stomach, colon, and at low levels in virgin breast, ovary, lung,small intestine and thymus. In contrast to PLM, Mat-8 is not expressedin liver, heart or skeletal muscle, which suggests distinct cellularfunctions for the two molecules (Morrison B W et al (1995) J Biol Chem270:2176-2182).

The extracellular and transmembrane domains of Mat-8 are homologous tothose of PLM. However, the cytoplasmic domain of Mat-8 is unrelated toPLM and contains no consensus phosphorylation sites for PKC or cAMPK.Expression of Mat-8 in Xenopus oocytes induces voltage-activatedchloride currents similar to those induced by expression of PLM(Morrison et al (1995), supra). However, direct ion channel formation byMat-8 has not been reported. The ability of Mat-8 protein to inducechloride channel activity, together with its tissue distribution(discussed above), suggests that this protein may be involved in theregulation of transepithelial transport in tissues containing absorptiveor secretory epithelia.

Additional proteins similar in structure to PLM and Mat-8 have beenfound to induce ion channel activity when expressed in Xenopus oocytes.Channel inducing factor (CHIF), found in colon and kidney, consists of asingle transmembrane domain and exhibits 50% sequence similarity to PLM(Attali B et al (1995) Proc Natl Acad Sci U.S.A. 92: 6092-6096). Xenopusoocytes injected with CHIF mRNA exhibit K+ specific channel activity.Slow-activating voltage dependent potassium ion channel (IsK; Takumi Tet al (1988) Science 242:1042-1045) is a single transmembrane domainglycoprotein present in epithelial cells, heart, uterus and lymphocytes(Attali B et al (1993) Nature 365:850-852). IsK induces both K+ and Cl-currents when expressed in Xenopus oocytes and HEK 293 cells. Theaccumulated evidence suggests that CHIF and IsK act as regulatorysubunits of pre-existing channel complexes rather than as channels perse (Attali B et al (1995), supra; Ben-Efraim I et al (1996) J Biol Chem271:8768-8771).

The sodium potassium ATPase (Na,K-ATPase) γ-subunit, formerly known asthe Na,K-ATPase proteolipid, is a small membrane protein thatco-purifies with the α- and β-subunits of Na,K-ATPase (Mercer R W et al(1993) J Cell Biol 121:579-586). The γ-subunit is a small membraneprotein consisting of 58 amino acids with a single transmembrane domain.This transmembrane domain is structurally related to the transmembranedomains of other PLM-like proteins. The γ-subunit may act as a regulatorof the ATP-dependent ion channel activity of Na,K-ATPase.

Discovery and molecular characterization of new members of the family ofPLM-like proteins satisfies a need in the art by providing newopportunities to understand and modulate physiological processesincluding neurotransmitter release, transepithelial transport, membranepotential stabilization, signal transduction and cell volume regulation.

SUMMARY OF THE INVENTION

The present invention features a novel human PLM-like protein,hereinafter referred to as HPLP, having chemical and structural homologyto PLM, Mat-8, CHIF, and Na,K-ATPase γ-subunit. Accordingly, theinvention features a substantially purified HPLP, encoded by amino acidsequence of SEQ ID NO:1, having structural characteristics of the familyof PLM-like proteins.

One aspect of the invention features isolated and substantially purifiedpolynucleotides which encode HPLP. In a particular aspect, thepolynucleotide is the nucleotide sequence of SEQ ID NO:2. In addition,the invention features nucleotide sequences which hybridize understringent conditions to SEQ ID NO:2.

The invention further relates to nucleic acid sequence encoding HPLP,oligonucleotides, peptide nucleic acids (PNA), fragments, portions orantisense molecules thereof. The present invention also relates to anexpression vector which includes polynucleotide encoding HPLP, its useto transform host cells or organisms and methods for producing theprotein. The invention also relates to antibodies which bindspecifically to HPLP and to a pharmaceutical composition comprisingsubstantially purified HPLP.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 shows the amino acid (SEQ ID NO:1) and nucleic acid sequences(SEQ ID NO:2) of the novel HPLP of the present invention produced usingMACDNASIS software (Hitachi Software Engineering Co Ltd, San BrunoCalif.).

FIG. 2 shows the amino acid sequence alignments among HPLP (SEQ IDNO:1), canine PLM (SEQ ID NO:3, GI 108084, Palmer et al, supra), humanMAT-8 (SEQ ID NO:4, GI 1085026, Morrison et al, supra), rat CHIF (SEQ IDNO:5, GI 951423, Attali B et al (1995), supra), and mouse Na,K-ATPaseγ-subunit (SEQ ID NO:6, GI 51112, Mercer R W et al, supra). Sequenceswere aligned using the multisequence alignment program of DNASTAR,software (DNAStar Inc, Madison Wis.).

FIG. 3 shows the hydrophobicity plot (generated using MACDNASISsoftware) for HPLP, SEQ ID NO:1; the X axis reflects amino acidposition, and the negative Y axis, hydrophobicity.

DESCRIPTION OF THE INVENTION

Definitions

"Nucleic acid sequence" as used herein refers to an oligonucleotide,nucleotide or polynucleotide, and fragments or portions thereof, and toDNA or RNA of genomic or synthetic origin which may be single- ordouble-stranded, and represent the sense or antisense strand. Similarly,amino acid sequence as used herein refers to peptide or proteinsequence.

"Consensus" as used herein may refer to a nucleic acid sequence 1) whichhas been resequenced to resolve uncalled bases, 2) which has beenextended using XL-PCR (Perkin Elmer, Norwalk Conn.) in the 5' and/or the3' direction and resequenced, 3) which has been assembled from theoverlapping sequences of more than one Incyte clone GCG FragmentAssembly System, (GCG, Madison Wis.), or 4) which has been both extendedand assembled.

"Peptide nucleic acid" as used herein refers to a molecule whichcomprises an oligomer to which an amino acid residue, such as lysine,and an amino group have been added. These small molecules, alsodesignated anti-gene agents, stop transcript elongation by binding totheir complementary (template) strand of nucleic acid (Nielsen PE et al(1993) Anticancer Drug Des 8:53-63).

A "deletion" is defined as a change in either nucleotide or amino acidsequence in which one or more nucleotides or amino acid residues,respectively, are absent.

An "insertion" or "addition" is that change in a nucleotide or aminoacid sequence which has resulted in the addition of one or morenucleotides or amino acid residues, respectively, as compared to thenaturally occurring HPLP.

A "substitution" results from the replacement of one or more nucleotidesor amino acids by different nucleotides or amino acids, respectively.

As used herein, HPLP refers to the amino acid sequence of substantiallypurified HPLP obtained from any species, particularly mammalian,including bovine, ovine, porcine, murine, equine, and preferably human,from any source whether natural, synthetic, semi-synthetic orrecombinant.

A "variant" of HPLP is defined as an amino acid sequence that differs byone or more amino acids. The variant may have "conservative" changes,wherein a substituted amino acid has similar structural or chemicalproperties, eg, replacement of leucine with isoleucine. More rarely, avariant may have "nonconservative" changes, eg, replacement of a glycinewith a tryptophan. Similar minor variations may also include amino aciddeletions or insertions, or both. Guidance in determining which and howmany amino acid residues may be substituted, inserted or deleted withoutabolishing biological or immunological activity may be found usingcomputer programs well known in the art, for example, DNAStar software.

The term "biologically active" refers to HPLP having structural,regulatory or biochemical functions of a naturally occurring HPLP.Likewise, "immunologically active" defines the capability of thenatural, recombinant or synthetic HPLP, or any oligopeptide thereof, toinduce a specific immune response in appropriate animals or cells and tobind with specific antibodies.

The term "agonist" refers to a molecule which, when bound to HPLP,causes a change in HPLP which modulates the biological activity of HPLP.

The term "antagonist" refers to a molecule which, when bound to HPLP,blocks the binding of an agonist to HPLP, which prevents theagonist-induced change in the biological activity of HPLP. Agonists andantagonists may include proteins, nucleic acids, carbohydrates, or othermolecules which bind to HPLP.

The term "modulate" as used herein refers to a change or an alterationin the biological activity of HPLP. Modulation may be an increase or adecrease in biological activity, a change in binding characteristics, orany other change in the biological properties of HPLP.

The term "derivative" as used herein refers to the chemical modificationof a nucleic acid encoding HPLP or the encoded HPLP. Illustrative ofsuch modifications would be replacement of hydrogen by an alkyl, acyl,or amino group. A nucleic acid derivative would encode a polypeptidewhich retains essential biological characteristics of natural HPLP.

As used herein, the term "substantially purified" refers to molecules,either nucleic or amino acid sequences, that are removed from theirnatural environment, isolated or separated, and are at least 60% free,preferably 75% free, and most preferably 90% free from other componentswith which they are naturally associated.

The term "hybridization" as used herein shall include "any process bywhich a strand of nucleic acid joins with a complementary strand throughbase pairing" (Coombs J (1994) Dictionary of Biotechnology, StocktonPress, New York N.Y.). Amplification is defined as the production ofadditional copies of a nucleic acid sequence and is generally carriedout using polymerase chain reaction technologies well known in the art(Dieffenbach C W and G S Dveksler (1995) PCR Primer, a LaboratoryManual, Cold Spring Harbor Press, Plainview N.Y.).

"Stringency" typically occurs in a range from about Tm-5° C. (5° C.below the Tm of the probe) to about 20° C. to 25° C. below Tm. As willbe understood by those of skill in the art, stringent hybridization canbe used to identify or detect identical polynucleotide sequences or toidentify or detect similar or related polynucleotide sequences.

Preferred Embodiments

The present invention relates to a novel human phospholemman-likeprotein (HPLP) originally identified among the cDNAs from a libraryconstructed from a human neuronal teratocarcinoma cell line and to theuse of the nucleic acid and amino acid sequences in the study,diagnosis, prevention and treatment of disease. The consensus nucleotidesequence, disclosed herein, was derived from the following overlappingand/or extended nucleic acid sequences: 97397 (PITUNOR01), 256782 and260077 (HNT2RAT01), 261205 and 261745 (HNT2AGT01), 397907 (PITUNOT02),555327 (SCORNOT01), 658934 (BRAINOT03), 691325 and 691710 (LUNGTUT02),743664, 744824, 749657, and 753284 (BRAITUT01), and 920208 and 920330(RATRNOT02).

Northern analysis using the LIFESEQ database (Incyte Pharmaceuticals,Palo Alto Calif.) shows that mRNA encoding HPLP is abundant in neuronaltissues (brain, neuronal cell lines and spinal cord). HPLP mRNA is alsofound in pituitary gland, heart, and lung. It must be noted thatnaturally occurring expression of HPLP is not necessarily limited tothese cells and tissue.

The present invention also encompasses HPLP variants. A preferredvariant is one having at least 80% amino acid sequence similarity to theamino acid sequence (SEQ ID NO:1), a more preferred HPLP variant is onehaving at least 90% amino acid sequence similarity to SEQ ID NO:1 and amost preferred HPLP variant is one having at least 95% amino acidsequence similarity to SEQ ID NO:1.

Nucleic acid encoding the human HPLP of the present invention was firstidentified in cDNA, Incyte Clone 261205 (SEQ ID NO:2), through acomputer-generated search for amino acid sequence alignments. Thenucleic acid sequence, SEQ ID NO:2, encodes the amino acid sequence, SEQID NO1.

The present invention is based, in part, on the chemical and structuralhomology among HPLP, PLM (SEQ ID NO:3; GI 108084; Palmer C et al,supra), MAT-8 (SEQ ID NO:4; GI 1085026; Morrison B et al (1995), supra),CHIF (SEQ ID NO:5; GI 951423, Attali B et al (1995), supra), andNa,K-ATPase γ-subunit (SEQ ID NO:6, GI 51112, Mercer R W et al, supra).PLM, Mat-8, CHIF, and Na,K-ATPase γ-subunit have, respectively, 46%,31%, 25%, and 27% sequence identity to HPLP. The identity increaseswithin the transmembrane domains of these proteins; the transmembranedomains of PLM, Mat-8, CHIF, and Na,K-ATPase γ-subunit have,respectively, 60%, 45%, 40%, and 60% sequence identity with thepredicted transmembrane domain of HPLP.

The HPLP protein sequence consists of 95 amino acids. From the aminoacid sequence alignments (FIG. 2) and the hydrophobicity plot (FIG. 3),HPLP is translated as a pre-protein. The HPLP signal peptide ispredicted to extend from residue 1 to residue 21. A single transmembranedomain is predicted to extend from residues 39 to 58, which terminatesin a positively-charged membrane stop transfer sequence (RRCK) atresidues 59 to 62.

The HPLP Coding Sequences

The extended and assembled nucleic acid and deduced amino acid sequencesof HPLP are shown in FIG. 1. In accordance with the invention, anynucleic acid sequence which encodes HPLP can be used to generaterecombinant molecules which express HPLP. In a specific embodimentdescribed herein, a partial sequence encoding HPLP was first isolated asIncyte Clone 261205 from an hNT2 cell line cDNA library (HNT2AGT01).

It will be appreciated by those skilled in the art that as a result ofthe degeneracy of the genetic code, a multitude of HPLP-encodingnucleotide sequences, some bearing minimal homology to the nucleotidesequences of any known and naturally occurring gene may be produced. Theinvention contemplates each and every possible variation of nucleotidesequence that could be made by selecting combinations based on possiblecodon choices. These combinations are made in accordance with thestandard triplet genetic code as applied to the nucleotide sequenceencoding naturally occurring HPLP, and all such variations are to beconsidered as being specifically disclosed.

Although nucleotide sequences which encode HPLP and its variants arepreferably capable of hybridizing to the nucleotide sequence of thenaturally occurring sequence under appropriately selected conditions ofstringency, it may be advantageous to produce nucleotide sequencesencoding HPLP or its derivatives possessing a substantially differentcodon usage. Codons may be selected to increase the rate at whichexpression of the peptide occurs in a particular prokaryotic oreukaryotic expression host in accordance with the frequency with whichparticular codons are utilized by the host. Other reasons forsubstantially altering the nucleotide sequence encoding HPLP and itsderivatives without altering the encoded amino acid sequences includethe production of RNA transcripts having more desirable properties, suchas a greater half-life, than transcripts produced from the naturallyoccurring sequence.

A DNA sequence, or portions thereof, encoding HPLP or its derivative maybe produced entirely by synthetic chemistry. After synthesis, the genemay be inserted into any of the many available DNA vectors and cellsystems using reagents that are known in the art. Moreover, syntheticchemistry may be used to introduce mutations into a sequence encodingHPLP or any portion thereof.

Also included within the scope of the present invention arepolynucleotide sequences that are capable of hybridizing to thenucleotide sequence of SEQ ID NO:2 under various conditions.Hybridization conditions are based on the melting temperature (Tm) ofthe nucleic acid binding complex or probe, as taught in Berger andKimmel (1987, Guide to Molecular Cloning Techniques, Methods inEnzymology, Vol 152, Academic Press, San Diego Calif.) incorporatedherein by reference, and on the salt concentrations under which thesteps of the process are carried out.

Altered nucleic acid sequences encoding HPLP which may be used inaccordance with the invention include deletions, insertions orsubstitutions of different nucleotides resulting in a polynucleotidethat encodes the same or a functionally equivalent HPLP. The protein mayalso show deletions, insertions or substitutions of amino acid residueswhich produce a silent change and result in a functionally equivalentHPLP. Deliberate amino acid substitutions may be made on the basis ofsimilarity in polarity, charge, solubility, hydrophobicity,hydrophilicity, and/or the amphipathic nature of the residues as long asthe biological activity of HPLP is retained. For example, negativelycharged amino acids include aspartic acid and glutamic acid; positivelycharged amino acids include lysine and arginine; and amino acids withuncharged polar head groups having similar hydrophilicity values includeleucine, isoleucine, valine; glycine, alanine; asparagine, glutamine;serine, threonine phenylalanine, and tyrosine.

Included within the scope of the present invention are alleles encodingHPLP. As used herein, an "allele" or "allelic sequence" is analternative form of the nucleic acid sequence encoding HPLP. Allelesresult from a mutation, ie, a change in the nucleic acid sequence, andgenerally produce altered mRNAs or polypeptides whose structure orfunction may or may not be altered. Any given gene may have none, one ormany allelic forms. Common mutational changes which give rise to allelesare generally ascribed to natural deletions, additions or substitutionsof amino acids. Each of these types of changes may occur alone, or incombination with the others, one or more times in a given sequence.

Methods for DNA sequencing may be used which are well known in the artand employ such enzymes as the Klenow fragment of DNA polymerase I,SEQUENASE (U.S. Biochemical Corp, Cleveland Ohio)), Taq polymerase(Perkin Elmer), thermostable T7 polymerase (Amersham, Chicago Ill.), orcombinations of recombinant polymerases and proofreading exonucleasessuch as the ELONGASE Amplification System marketed by Gibco BRL(Gaithersburg Md.). Preferably, the process is automated with machinessuch as the Hamilton Micro Lab 2200 (Hamilton, Reno Nev.), PeltierThermal Cycler (PTC200; MJ Research, Watertown Mass.) and the ABI 377DNA sequencers (Perkin Elmer).

Extending the Polynucleotide Sequence

The polynucleotide sequence encoding HPLP may be extended utilizingpartial nucleotide sequence and various methods known in the art todetect upstream sequences such as promoters and regulatory elements. Forexample, Gobinda et al (1993; PCR Methods Applic 2:318-22) use"restriction-site" polymerase chain reaction (PCR) as a direct methodwhich uses universal primers to retrieve unknown sequence adjacent to aknown locus. First, genomic DNA is amplified in the presence of primerto a linker sequence and a primer specific to the known region. Theamplified sequences are subjected to a second round of PCR with the samelinker primer and another specific primer internal to the first one.Products of each round of PCR are transcribed with an appropriate RNApolymerase and sequenced using reverse transcriptase.

Inverse PCR can be used to amplify or extend sequences using divergentprimers based on a known region (Triglia T et al (1988) Nucleic AcidsRes 16:8186). The primers may be designed using OLIGO 4.06 PrimerAnalysis Software (1992; National Biosciences Inc, Plymouth Minn.), oranother appropriate program, to be 22-30 nucleotides in length, to havea GC content of 50% or more, and to anneal to the target sequence attemperatures about 68°-72° C. The method uses several restrictionenzymes to generate a suitable fragment in the known region of a gene.The fragment is then circularized by intramolecular ligation and used asa PCR template.

Capture PCR (Lagerstrom M et al (1991) PCR Methods Applic 1:111-19) is amethod for PCR amplification of DNA fragments adjacent to a knownsequence in human and yeast artificial chromosome DNA. Capture PCR alsorequires multiple restriction enzyme digestions and ligations to placean engineered double-stranded sequence into an unknown portion of theDNA molecule before PCR.

Another method which may be used to retrieve unknown sequence is walkingPCR (Parker J D et al (1991) Nucleic Acids Res 19:3055-60), whichinvolves targeted gene walking. Alternatively, PCR, nested primers,PROMOTERFINDER (Clontech, Palo Alto Calif.) and PROMOTERFINDER librariescan be used to walk in genomic DNA. This process avoids the need toscreen libraries and is useful in finding intron/exon junctions.

Preferred libraries for screening for full length cDNAs are those whichhave been size-selected to include larger cDNAs. Also, random primedlibraries are preferred in that they will contain more sequences whichcontain the 5' and upstream regions of genes. A randomly primed librarymay be particularly useful if an oligo d(T) library does not yield afull-length cDNA. Genomic libraries are useful for extension into the 5'nontranslated regulatory region.

Capillary electrophoresis may be used to analyze either the size orconfirm the nucleotide sequence in sequencing or PCR products. Systemsfor rapid sequencing are available from Perkin Elmer, BeckmanInstruments (Fullerton Calif.), and other companies. Capillarysequencing may employ flowable polymers for electrophoretic separation,four different fluorescent dyes (one for each nucleotide) which arelaser activated, and detection of the emitted wavelengths by a chargecoupled device camera. Output/light intensity is converted to electricalsignal using appropriate software (eg. GENOTYPER and SEQUENCES NAVIGATORfrom Perkin Elmer) and the entire process from loading of samples tocomputer analysis and electronic data display is computer controlled.Capillary electrophoresis is particularly suited to the sequencing ofsmall pieces of DNA which might be present in limited amounts in aparticular sample. The reproducible sequencing of up to 350 bp of M13phage DNA in 30 min has been reported (Ruiz-Martinez M C et al (1993)Anal Chem 65:2851-8).

Expression of the Nucleotide Sequence

In accordance with the present invention, polynucleotide sequences whichencode HPLP, fragments of the polypeptide, fusion proteins or functionalequivalents thereof may be used in recombinant DNA molecules that directthe expression of HPLP in appropriate host cells. Due to the inherentdegeneracy of the genetic code, other DNA sequences which encodesubstantially the same or a functionally equivalent amino acid sequence,may be used to clone and express HPLP. As will be understood by those ofskill in the art, it may be advantageous to produce HPLP-encodingnucleotide sequences possessing non-naturally occurring codons. Codonspreferred by a particular prokaryotic or eukaryotic host (Murray E et al(1989) Nuc Acids Res 17:477-508) can be selected, for example, toincrease the rate of HPLP expression or to produce recombinant RNAtranscripts having desirable properties, such as a longer half-life,than transcripts produced from naturally occurring sequence.

The nucleotide sequences of the present invention can be engineered inorder to alter HPLP-encoding sequence for a variety of reasons,including but not limited to, alterations which modify the cloning,processing and/or expression of the gene product. For example, mutationsmay be introduced using techniques which are well known in the art, eg,site-directed mutagenesis to insert new restriction sites, to alterglycosylation patterns, to change codon preference, to produce splicevariants, etc.

In another embodiment of the invention, a natural, modified orrecombinant HPLP-encoding sequence may be ligated to a heterologoussequence to encode a fusion protein. For example, for screening ofpeptide libraries for inhibitors of HPLP activity, it may be useful toencode a chimeric HPLP protein that is recognized by a commerciallyavailable antibody. A fusion protein may also be engineered to contain acleavage site located between HPLP and the heterologous proteinsequence, so that the HPLP may be cleaved and substantially purifiedaway from the heterologous moiety.

In an alternate embodiment of the invention, the sequence encoding HPLPmay be synthesized, whole or in part, using chemical methods well knownin the art (see Caruthers M H et al (1980) Nuc Acids Res Symp Ser215-23, Horn T et al(1980) Nuc Acids Res Symp Ser 225-32, etc).Alternatively, the protein itself may be produced using chemical methodsto synthesize an amino acid sequence for HPLP, whole or in part. Forexample, peptide synthesis can be performed using various solid-phasetechniques (Roberge J Y et al (1995) Science 269:202-204) and automatedsynthesis may be achieved, for example, using the ABI 431A PeptideSynthesizer (Perkin Elmer) in accordance with the instructions providedby the manufacturer.

The newly synthesized peptide can be substantially purified bypreparative high performance liquid chromatography (eg, Creighton (1983)Proteins, Structures and Molecular Principles, W H Freeman and Co, NewYork N.Y.). The composition of the synthetic peptides may be confirmedby amino acid analysis or sequencing (eg, the Edman degradationprocedure; Creighton, supra). Additionally the amino acid sequence ofHPLP, or any part thereof, may be altered during direct synthesis and/orcombined using chemical methods with sequences from other proteins, orany part thereof, to produce a variant polypeptide.

Expression Systems

In order to express a biologically active HPLP, the nucleotide sequenceencoding HPLP or its functional equivalent, is inserted into anappropriate expression vector, ie, a vector which contains the necessaryelements for the transcription and translation of the inserted codingsequence.

Methods which are well known to those skilled in the art can be used toconstruct expression vectors containing a sequence encoding HPLP andappropriate transcriptional or translational controls. These methodsinclude in vitro recombinant DNA techniques, synthetic techniques and invivo recombination or genetic recombination. Such techniques aredescribed in Sambrook et al (1989) Molecular Cloning, A LaboratoryManual, Cold Spring Harbor Press, Plainview N.Y. and Ausubel F M et al(1989) Current Protocols in Molecular Biology, John Wiley & Sons, NewYork N.Y.

A variety of expression vector/host systems may be utilized to containand express a sequence encoding HPLP. These include but are not limitedto microorganisms such as bacteria transformed with recombinantbacteriophage, plasmid or cosmid DNA expression vectors; yeasttransformed with yeast expression vectors; insect cell systems infectedwith virus expression vectors (eg, baculovirus); plant cell systemstransfected with virus expression vectors (eg, cauliflower mosaic virus,CaMV; tobacco mosaic virus, TMV) or transformed with bacterialexpression vectors (eg, Ti or pBR322 plasmid); or animal cell systems.

The "control elements" or "regulatory sequences" of these systems varyin their strength and specificities and are those nontranslated regionsof the vector, enhancers, promoters, and 3' and 5' untranslated regions,which interact with host cellular proteins to carry out transcriptionand translation. Depending on the vector system and host utilized, anynumber of suitable transcription and translation elements, includingconstitutive and inducible promoters, may be used. For example, whencloning in bacterial systems, inducible promoters such as the hybridlacZ promoter of the BLUESCRIPT phagemid (Stratagene, LaJolla Calif.) orPSPORT1 (Gibco BRL) and ptrp-lac hybrids and the like may be used. Thebaculovirus polyhedrin promoter may be used in insect cells. Promotersor enhancers derived from the genomes of plant cells (eg, heat shock,RUBISCO; and storage protein genes) or from plant viruses (eg, viralpromoters or leader sequences) may be cloned into the vector. Inmammalian cell systems, promoters from the mammalian genes or frommammalian viruses are most appropriate. If it is necessary to generate acell line that contains multiple copies of the sequence encoding HPLP,vectors based on SV40 or EBV may be used with an appropriate selectablemarker.

In bacterial systems, a number of expression vectors may be selecteddepending upon the use intended for HPLP. For example, when largequantities of HPLP are needed for the induction of antibodies, vectorswhich direct high level expression of fusion proteins that are readilypurified may be desirable. Such vectors include, but are not limited to,the multifunctional E. coli cloning and expression vectors such asBLUESCRIPT (Stratagene), in which the sequence encoding HPLP may beligated into the vector in frame with sequences for the amino-terminalMet and the subsequent 7 residues of β-galactosidase so that a hybridprotein is produced; pIN vectors (Van Heeke & Schuster (1989) J BiolChem 264:5503-5509); and the like. pGEX vectors (Promega, Madison Wis.)may also be used to express foreign polypeptides as fusion proteins withglutathione S-transferase (GST). In general, such fusion proteins aresoluble and can easily be purified from lysed cells by adsorption toglutathione-agarose beads followed by elution in the presence of freeglutathione. Proteins made in such systems are designed to includeheparin, thrombin or factor XA protease cleavage sites so that thecloned polypeptide of interest can be released from the GST moiety atwill.

In the yeast, Saccharomyces cerevisiae, a number of vectors containingconstitutive or inducible promoters such as alpha factor, alcoholoxidase and PGH may be used. For reviews, see Ausubel et al (supra) andGrant et al (1987) Methods in Enzymology 153:516-544.

In cases where plant expression vectors are used, the expression of asequence encoding HPLP may be driven by any of a number of promoters.For example, viral promoters such as the 35S and 19S promoters of CaMV(Brisson et al (1984) Nature 310:511-514) may be used alone or incombination with the omega leader sequence from TMV (Takamatsu et al(1987) EMBO J 6:307-311). Alternatively, plant promoters such as thesmall subunit of RUBISCO (Coruzzi et al (1984) EMBO J 3:1671-1680;Broglie et al (1984) Science 224:838-843); or heat shock promoters(Winter J and Sinibaldi R M (1991) Results Probl Cell Differ 17:85-105)may be used. These constructs can be introduced into plant cells bydirect DNA transformation or pathogen-mediated transfection. For reviewsof such techniques, see Hobbs S or Murry L E in McGraw Hill Yearbook ofScience and Technology (1992) McGraw Hill New York N.Y., pp 191-196 orWeissbach and Weissbach (1988) Methods for Plant Molecular Biology,Academic Press, New York N.Y., pp 421-463.

An alternative expression system which may be used to express HPLP is aninsect system. In one such system, Autographa californica nuclearpolyhedrosis virus (AcNPV) is used as a vector to express foreign genesin Spodoptera frugiperda cells or in Trichoplusia larvae. The sequenceencoding HPLP may be cloned into a nonessential region of the virus,such as the polyhedrin gene, and placed under control of the polyhedrinpromoter. Successful insertion of the sequence encoding HPLP will renderthe polyhedrin gene inactive and produce recombinant virus lacking coatprotein. The recombinant viruses are then used to infect S. frugiperdacells or Trichoplusia larvae in which HPLP is expressed (Smith et al(1983) J Virol 46:584; Engelhard E K et al (1994) Proc Nat Acad Sci91:3224-7).

In mammalian host cells, a number of viral-based expression systems maybe utilized. In cases where an adenovirus is used as an expressionvector, a sequence encoding HPLP may be ligated into an adenovirustranscription/translation complex consisting of the late promoter andtripartite leader sequence. Insertion in a nonessential E1 or E3 regionof the viral genome will result in a viable virus capable of expressingin infected host cells (Logan and Shenk (1984) Proc Natl Acad Sci81:3655-59). In addition, transcription enhancers, such as the roussarcoma virus (RSV) enhancer, may be used to increase expression inmammalian host cells.

Specific initiation signals may also be required for efficienttranslation of a sequence encoding HPLP. These signals include the ATGinitiation codon and adjacent sequences. In cases where the sequenceencoding HPLP, its initiation codon and upstream sequences are insertedinto the most appropriate expression vector, no additional translationalcontrol signals may be needed. However, in cases where only codingsequence, or a portion thereof, is inserted, exogenous translationalcontrol signals including the ATG initiation codon must be provided.Furthermore, the initiation codon must be in the correct reading frameto ensure translation of the entire insert. Exogenous translationalelements and initiation codons can be of various origins, both naturaland synthetic. The efficiency of expression may be enhanced by theinclusion of enhancers appropriate to the cell system in use (Scharf Det al (1994) Results Probl Cell Differ 20:125-62; Bittner et al (1987)Methods in Enzymol 153:516-544).

In addition, a host cell strain may be chosen for its ability tomodulate the expression of the inserted sequences or to process theexpressed protein in the desired fashion. Such modifications of thepolypeptide include, but are not limited to, acetylation, carboxylation,glycosylation, phosphorylation, lipidation and acylation.Post-translational processing which cleaves a "prepro" form of theprotein may also be important for correct insertion, folding and/orfunction. Different host cells such as CHO, HeLa, MDCK, 293, WI38, etchave specific cellular machinery and characteristic mechanisms for suchpost-translational activities and may be chosen to ensure the correctmodification and processing of the introduced, foreign protein.

For long-term, high-yield production of recombinant proteins, stableexpression is preferred. For example, cell lines which stably expressHPLP may be transformed using expression vectors which contain viralorigins of replication or endogenous expression elements and aselectable marker gene. Following the introduction of the vector, cellsmay be allowed to grow for 1-2 days in an enriched media before they areswitched to selective media. The purpose of the selectable marker is toconfer resistance to selection, and its presence allows growth andrecovery of cells which successfully express the introduced sequences.Resistant clumps of stably transformed cells can be proliferated usingtissue culture techniques appropriate to the cell type.

Any number of selection systems may be used to recover transformed celllines. These include, but are not limited to, the herpes simplex virusthymidine kinase (Wigler M et al (1977) Cell 11:223-32) and adeninephosphoribosyltransferase (Lowy I et al (1980) Cell 22:817-23) geneswhich can be employed in tk- or aprt- cells, respectively. Also,antimetabolite, antibiotic or herbicide resistance can be used as thebasis for selection; for example, dhfr which confers resistance tomethotrexate (Wigler M et al (1980) Proc Natl Acad Sci 77:3567-70); npt,which confers resistance to the aminoglycosides, neomycin and G-418(Colbere-Garapin F et al (1981) J Mol Biol 150:1-14), and als or pat,which confer resistance to chlorsulfuron and phosphinotricinacetyltransferase, respectively (Murry, supra). Additional selectablegenes have been described, for example, trpB, which allows cells toutilize indole in place of tryptophan, or hisD, which allows cells toutilize histinol in place of histidine (Hartman S C and R C Mulligan(1988) Proc NatI Acad Sci 85:8047-51). Recently, the use of visiblemarkers has gained popularity with such markers as anthocyanins,βglucuronidase and its substrate, GUS, and luciferase and its substrate,luciferin, being widely used not only to identify transformants, butalso to quantify the amount of transient or stable protein expressionattributable to a specific vector system (Rhodes C A et al (1995)Methods Mol Biol 55:121-131).

Identification of Transformants Containing the Polynucleotide Sequence

Although the presence/absence of marker gene expression suggests thatthe gene of interest is also present, its presence and expression shouldbe confirmed. For example, if the sequence encoding HPLP is insertedwithin a marker gene sequence, recombinant cells containing the sequenceencoding HPLP can be identified by the absence of marker gene function.Alternatively, a marker gene can be placed in tandem with the sequenceencoding HPLP under the control of a single promoter. Expression of themarker gene in response to induction or selection usually indicatesexpression of the tandem sequence as well.

Alternatively, host cells which contain the sequence encoding HPLP andexpressing HPLP may be identified by a variety of procedures known tothose of skill in the art. These procedures include, but are not limitedto, DNA-DNA or DNA-RNA hybridization and protein bioassay or immunoassaytechniques which include membrane, solution, or chip based technologiesfor the detection and/or quantification of the nucleic acid or protein.

The presence of the polynucleotide sequence encoding HPLP can bedetected by DNA-DNA or DNA-RNA hybridization or amplification usingprobes, portions or fragments of the sequence encoding HPLP. Nucleicacid amplification based assays involve the use of oligonucleotides oroligomers based on the nucleic acid sequence to detect transformantscontaining DNA or RNA encoding HPLP. As used herein "oligonucleotides"or "oligomers" refer to a nucleic acid sequence of at least about 10nucleotides and as many as about 60 nucleotides, preferably about 15 to30 nucleotides, and more preferably about 20-25 nucleotides which can beused as a probe or amplimer. A variety of protocols for detecting andmeasuring the expression of HPLP, using either polyclonal or monoclonalantibodies specific for the protein are known in the art. Examplesinclude enzyme-linked immunosorbent assay (ELISA), radioimmunoassay(RIA) and fluorescent activated cell sorting (FACS). A two-site,monoclonal-based immunoassay utilizing monoclonal antibodies reactive totwo non-interfering epitopes on HPLP is preferred, but a competitivebinding assay may be employed. These and other assays are described,among other places, in Hampton R et al (1990, Serological Methods, aLaboratory Manual, APS Press, St Paul Min.) and Maddox D E et al (1983,J Exp Med 158:1211).

A wide variety of labels and conjugation techniques are known by thoseskilled in the art and can be used in various nucleic acid and aminoacid assays. Means for producing labeled hybridization or PCR probes fordetecting related sequences include oligolabeling, nick translation,end-labeling or PCR amplification using a labeled nucleotide.Alternatively, the HPLP-encoding sequence, or any portion of it, may becloned into a vector for the production of an mRNA probe. Such vectorsare known in the art, are commercially available, and may be used tosynthesize RNA probes in vitro by addition of an appropriate RNApolymerase such as T7, T3 or SP6 and labeled nucleotides.

A number of companies such as Pharmacia Biotech (Piscataway N.J.),Promega (Madison Wis.), and U.S. Biochemical Corp (Cleveland Ohio)supply commercial kits and protocols for these procedures. Suitablereporter molecules or labels include those radionuclides, enzymes,fluorescent, chemiluminescent, or chromogenic agents as well assubstrates, cofactors, inhibitors, magnetic particles and the like.Patents teaching the use of such labels include U.S. Pat. Nos.3,817,837; 3,850,752; 3,939,350; 3,996,345; 4,277,437; 4,275,149 and4,366,241. Also, recombinant immunoglobulins may be produced as shown inU.S. Pat. No. 4,816,567 incorporated herein by reference.

Purification of HPLP

Host cells transformed with a nucleotide sequence encoding HPLP may becultured under conditions suitable for the expression and recovery ofthe encoded protein from cell culture. The protein produced by arecombinant cell may be secreted or contained intracellularly dependingon the sequence and/or the vector used. As will be understood by thoseof skill in the art, expression vectors containing sequence encodingHPLP can be designed with signal sequences which direct secretion ofHPLP through a prokaryotic or eukaryotic cell membrane. Otherrecombinant constructions may join the sequence encoding HPLP tonucleotide sequence encoding a polypeptide domain which will facilitatepurification of soluble proteins (Kroll D J et al (1993) DNA Cell Biol12:441-53; of discussion of vectors infra containing fusion proteins).

HPLP may also be expressed as a recombinant protein with one or moreadditional polypeptide domains added to facilitate protein purification.Such purification facilitating domains include, but are not limited to,metal chelating peptides such as histidine-tryptophan modules that allowpurification on immobilized metals, protein A domains that allowpurification on immobilized immunoglobulin, and the domain utilized inthe FLAGS extension/affinity purification system (Immunex Corp, SeattleWash.). The inclusion of a cleavable linker sequences such as Factor XAor enterokinase (Invitrogen, San Diego Calif.) between the purificationdomain and HPLP is useful to facilitate purification. One suchexpression vector provides for expression of a fusion protein comprisingthe sequence encoding HPLP and nucleic acid sequence encoding 6histidine residues followed by thioredoxin and an enterokinase cleavagesite. The histidine residues facilitate purification while theenterokinase cleavage site provides a means for purifying HPLP from thefusion protein.

In addition to recombinant production, fragments of HPLP may be producedby direct peptide synthesis using solid-phase techniques (cf Stewart etal (1969) Solid-Phase Peptide Synthesis, W H Freeman Co, San Francisco;Merrifield J (1963) J Am Chem Soc 85:2149-2154). In vitro proteinsynthesis may be performed using manual techniques or by automation.Automated synthesis may be achieved, for example, using AppliedBiosystems 431A Peptide Synthesizer (Perkin Elmer, Foster City Calif.)in accordance with the instructions provided by the manufacturer.Various fragments of HPLP may be chemically synthesized separately andcombined using chemical methods to produce the full length molecule.

Uses of HPLP

The rationale for diagnostic and therapeutic uses of sequences encodingHPLP is based on the nucleotide and amino acid sequences, their homologyto PLM-like transmembrane proteins associated with ion transport, theirtissue distribution primarily in brain and nervous system tissues, butalso in pituitary, heart, and lung, and the known associations andfunctions of PLM-like transmembrane proteins.

HPLP may modify or regulate ion currents generated in the central and/orautonomic nervous system. Therefore, mutations in or altered expressionof HPLP may be associated with diseases and conditions relating todefective ion transport. Such disorders may include, but are not limitedto, defects in nerve signal transmission, membrane potential generation,or fluid volume regulation, such as Alzheimer's disease, Parkinson'sdisease, Huntington's disease, amyotrophic lateral sclerosis, andhydrocephalus.

HPLP or its fragments may be used to identify specific molecules thatmodulate the activity of HPLP, such as agonists, antagonists orinhibitors. Furthermore, HPLP is useful as an investigative tool in thestudy of the control of ion transport and membrane potential in bothnormal and diseased cells.

HPLP-specific antibodies are useful for the diagnosis of conditions anddiseases associated with expression of the polypeptides. Antibodiesspecifically recognizing HPLP may be used to quantitate HPLP fordiagnostic purposes. A diagnostic test for altered expression of HPLPmay accelerate diagnosis and proper treatment of conditions associatedwith HPLP.

The HPLP nucleic acid sequence of SEQ ID NO:2 can be incorporated intoeffective eukaryotic expression vectors and directly administered intosomatic cells for gene therapy. In like manner, RNA transcripts producedin vitro may be encapsulated in and administered via liposomes. Suchvectors and transcripts may function transiently or may be incorporatedinto the host chromosomal DNA for longer term expression.

In vivo delivery of genetic constructs into subjects is developed to thepoint of targeting specific cell types. The delivery to specific cellshas been accomplished, for instance, by complexing nucleic acids withproteinous ligands that recognize cell specific receptors which mediateuptake (cf Wu GY et al (1991) J Biol Chem 266:14338-42). Alternatively,recombinant nucleic acid constructs may be injected directly for localuptake and integration (Jiao S et al (1992) Human Gene Therapy 3:21-33).

HPLP Antibodies

HPLP-specific antibodies are useful for the diagnosis and treatment ofconditions and diseases associated with expression of HPLP. Suchantibodies include, but are not limited to, polyclonal, monoclonal,chimeric, single chain, Fab fragments and fragments produced by a Fabexpression library. Neutralizing antibodies, ie, those which inhibitdimer formation, are especially preferred for diagnostics andtherapeutics.

It is not necessary that the portion of HPLP used for antibody inductionhave biological activity; however, the protein fragment, or oligopeptidemust be antigenic. Peptides used to induce specific antibodies may havean amino acid sequence consisting of at least five amino acids, andpreferably at least 10 amino acids. Preferably, they should mimic aportion of the amino acid sequence of the natural protein and maycontain the entire amino acid sequence of a small, naturally occurringmolecule. Short stretches of HPLP amino acids may be fused with those ofanother protein such as keyhole limpet hemocyanin and antibody producedagainst the chimeric molecule. Procedures well known in the art can beused for the production of antibodies to HPLP.

For the production of antibodies, various hosts including goats,rabbits, rats, mice, etc may be immunized by injection with HPLP or anyportion, fragment or oligopeptide which retains immunogenic properties.Depending on the host species, various adjuvants may be used to increaseimmunological response. Such adjuvants include but are not limited toFreund's, mineral gels such as aluminum hydroxide, and surface activesubstances such as lysolecithin, pluronic polyols, polyanions, peptides,oil emulsions, keyhole limpet hemocyanin, and dinitrophenol. BCG(bacilli Calmette-Guerin) and Corynebacterium parvum are potentiallyuseful human adjuvants.

Monoclonal antibodies to HPLP may be prepared using any technique whichprovides for the production of antibody molecules by continuous celllines in culture. These include but are not limited to the hybridomatechnique originally described by Koehler and Milstein (1975 Nature256:495-497), the human B-cell hybridoma technique (Kosbor et al (1983)Immunol Today 4:72; Cote et al (1983) Proc Natl Acad Sci 80:2026-2030)and the EBV-hybridoma technique (Cole et al (1985) Monoclonal Antibodiesand Cancer Therapy, Alan R Liss Inc, New York N.Y., pp 77-96).

In addition, techniques developed for the production of "chimericantibodies", the splicing of mouse antibody genes to human antibodygenes to obtain a molecule with appropriate antigen specificity andbiological activity can be used (Morrison et al (1984) Proc Natl AcadSci 81:6851-6855; Neuberger et al (1984) Nature 312:604-608; Takeda etal (1985) Nature 314:452-454). Alternatively, techniques described forthe production of single chain antibodies (U.S. Pat. No. 4,946,778) canbe adapted to produce HPLP-specific single chain antibodies.

Antibodies may also be produced by inducing in vivo production in thelymphocyte population or by screening recombinant immunoglobulinlibraries or panels of highly specific binding reagents as disclosed inOrlandi et al (1989, Proc Natl Acad Sci 86: 3833-3837), and Winter G andMilstein C (1991; Nature 349:293-299).

Antibody fragments which contain specific binding sites for HPLP mayalso be generated. For example, such fragments include, but are notlimited to, the F(ab')2 fragments which can be produced by pepsindigestion of the antibody molecule and the Fab fragments which can begenerated by reducing the disulfide bridges of the F(ab')2 fragments.Alternatively, Fab expression libraries may be constructed to allowrapid and easy identification of monoclonal Fab fragments with thedesired specificity (Huse W D et al (1989) Science 256:1275-1281).

A variety of protocols for competitive binding or immunoradiometricassays using either polyclonal or monoclonal antibodies with establishedspecificities are well known in the art. Such immunoassays typicallyinvolve the formation of complexes between HPLP and its specificantibody and the measurement of complex formation. A two-site,monoclonal-based immunoassay utilizing monoclonal antibodies reactive totwo noninterfering epitopes on a specific HPLP protein is preferred, buta competitive binding assay may also be employed. These assays aredescribed in Maddox D E et al (1983, J Exp Med 158:1211).

Diagnostic Assays Using HPLP Specific Antibodies

Particular HPLP antibodies are useful for the diagnosis of conditions ordiseases characterized by expression of HPLP or in assays to monitorpatients being treated with HPLP, its fragments, agonists or inhibitors.Diagnostic assays for HPLP include methods utilizing the antibody and alabel to detect HPLP in human body fluids or extracts of cells ortissues. The polypeptides and antibodies of the present invention may beused with or without modification. Frequently, the polypeptides andantibodies will be labeled by joining them, either covalently ornoncovalently, with a reporter molecule. A wide variety of reportermolecules are known, several of which were described above.

A variety of protocols for measuring HPLP, using either polyclonal ormonoclonal antibodies specific for the respective protein are known inthe art. Examples include enzyme-linked immunosorbent assay (ELISA),radioimmunoassay (RIA) and fluorescent activated cell sorting (FACS). Atwo-site, monoclonal-based immunoassay utilizing monoclonal antibodiesreactive to two non-interfering epitopes on HPLP is preferred, but acompetitive binding assay may be employed. These assays are described,among other places, in Maddox, D E et al (1983) J Exp Med 158:1211.

In order to provide a basis for diagnosis, normal or standard values forHPLP expression must be established. This is accomplished by combiningbody fluids or cell extracts taken from normal subjects, either animalor human, with antibody to HPLP under conditions suitable for complexformation which are well known in the art. The amount of standardcomplex formation may be quantified by comparing various artificialmembranes containing known quantities of HPLP with both control anddisease samples from biopsied tissues. Then, standard values obtainedfrom normal samples may be compared with values obtained from samplesfrom subjects potentially affected by disease. Deviation betweenstandard and subject values establishes the presence of disease state.

Drug Screening

HPLP, its catalytic or immunogenic fragments or oligopeptides thereof,can be used for screening therapeutic compounds in any of a variety ofdrug screening techniques. The fragment employed in such a test may befree in solution, affixed to a solid support, borne on a cell surface,or located intracellularly. The formation of binding complexes, betweenHPLP and the agent being tested, may be measured.

Another technique for drug screening which may be used for highthroughput screening of compounds having suitable binding affinity tothe HPLP is described in detail in "Determination of Amino Acid SequenceAntigenicity" by Geysen H N, WO Application 84/03564, published on Sep.13, 1984, and incorporated herein by reference. In summary, largenumbers of different small peptide test compounds are synthesized on asolid substrate, such as plastic pins or some other surface. The peptidetest compounds are reacted with fragments of HPLP and washed. Bound HPLPis then detected by methods well known in the art. Substantiallypurified HPLP can also be coated directly onto plates for use in theaforementioned drug screening techniques. Alternatively,non-neutralizing antibodies can be used to capture the peptide andimmobilize it on a solid support.

This invention also contemplates the use of competitive drug screeningassays in which neutralizing antibodies capable of binding HPLPspecifically compete with a test compound for binding HPLP. In thismanner, the antibodies can be used to detect the presence of any peptidewhich shares one or more antigenic determinants with HPLP.

Uses of the Polynucleotide Encoding HPLP

A polynucleotide sequence encoding HPLP or any part thereof may be usedfor diagnostic and/or therapeutic purposes. For diagnostic purposes, thesequence encoding HPLP of this invention may be used to detect andquantitate gene expression in biopsied tissues in which HPLP may beexpressed in response to oncogenes. The diagnostic assay is useful todistinguish between absence, presence, and excess expression of HPLP andto monitor regulation of HPLP levels during therapeutic intervention.Included in the scope of the invention are oligonucleotide sequences,antisense RNA and DNA molecules, and peptide nucleic acids (PNA).

Another aspect of the subject invention is to provide for hybridizationor PCR probes which are capable of detecting polynucleotide sequences,including genomic sequences, encoding HPLP or closely related molecules.The specificity of the probe, whether it is made from a highly specificregion, eg, 10 unique nucleotides in the 5' regulatory region, or a lessspecific region, eg, especially in the 3' region, and the stringency ofthe hybridization or amplification (maximal, high, intermediate or low)will determine whether the probe identifies only naturally occurringHPLP, alleles or related sequences.

Probes may also be used for the detection of related sequences andshould preferably contain at least 50% of the nucleotides from any ofthese sequences encoding HPLP. The hybridization probes of the subjectinvention may be derived from the nucleotide sequence of SEQ ID NO:2 orfrom genomic sequence including promoter, enhancer elements and intronsof the naturally occurring sequence encoding HPLP. Hybridization probesmay be labeled by a variety of reporter groups, including radionuclidessuch as 32P or 35S, or enzymatic labels such as alkaline phosphatasecoupled to the probe via avidin/biotin coupling systems, and the like.

Other means for producing specific hybridization probes for DNAs includethe cloning of nucleic acid sequences encoding HPLP or HPLP derivativesinto vectors for the production of mRNA probes. Such vectors are knownin the art and are commercially available and may be used to synthesizeRNA probes in vitro by means of the addition of the appropriate RNApolymerase as T7 or SP6 RNA polymerase and the appropriate radioactivelylabeled nucleotides.

Diagnostic Use

Polynucleotide sequences encoding HPLP may be used for the diagnosis ofconditions or diseases with which the expression of HPLP is associated.For example, polynucleotide sequences encoding HPLP may be used inhybridization or PCR assays of fluids or tissues from biopsies to detectHPLP expression. The form of such qualitative or quantitative methodsmay include Southern or northern analysis, dot blot or othermembrane-based technologies; PCR technologies; dip stick, pin, chip andELISA technologies. All of these techniques are well known in the artand are the basis of many commercially available diagnostic kits.

The HPLP-encoding nucleotide sequences disclosed herein provide thebasis for assays that detect activation or induction associated withinflammation or disease. The nucleotide sequence may be labeled bymethods known in the art and added to a fluid or tissue sample from apatient under conditions suitable for the formation of hybridizationcomplexes. After an incubation period, the sample is washed with acompatible fluid which optionally contains a dye (or other labelrequiring a developer) if the nucleotide has been labeled with anenzyme. After the compatible fluid is rinsed off, the dye is quantitatedand compared with a standard. If the amount of dye in the biopsied orextracted sample is significantly elevated over that of a comparablecontrol sample, the nucleotide sequence has hybridized with nucleotidesequences in the sample, and the presence of elevated levels ofnucleotide sequences encoding HPLP in the sample indicates the presenceof the associated inflammation and/or disease.

Such assays may also be used to evaluate the efficacy of a particulartherapeutic treatment regime in animal studies, in clinical trials, orin monitoring the treatment of an individual patient. In order toprovide a basis for the diagnosis of disease, a normal or standardprofile for HPLP expression must be established. This is accomplished bycombining body fluids or cell extracts taken from normal subjects,either animal or human, with HPLP, or a portion thereof, underconditions suitable for hybridization or amplification. Standardhybridization may be quantified by comparing the values obtained fornormal subjects with a dilution series of HPLP run in the sameexperiment where a known amount of substantially purified HPLP is used.Standard values obtained from normal samples may be compared with valuesobtained from samples from patients affected by HPLP-associateddiseases. Deviation between standard and subject values establishes thepresence of disease.

Once disease is established, a therapeutic agent is administered and atreatment profile is generated. Such assays may be repeated on a regularbasis to evaluate whether the values in the profile progress toward orreturn to the normal or standard pattern. Successive treatment profilesmay be used to show the efficacy of treatment over a period of severaldays or several months.

PCR, may be used as described in U.S. Pat. Nos. 4,683,195 and 4,965,188and provides additional uses for oligonucleotides based upon thesequence encoding HELP. Such oligomers are generally chemicallysynthesized, but they may be generated enzymatically or produced from arecombinant source. Oligomers generally comprise two nucleotidesequences, one with sense orientation (5'→3') and one with antisense(3'→5'), employed under optimized conditions for identification of aspecific gene or condition. The same two oligomers, nested sets ofoligomers, or even a degenerate pool of oligomers may be employed underless stringent conditions for detection and/or quantitation of closelyrelated DNA or RNA sequences.

Additionally, methods which may be used to quantitate the expression ofa particular molecule include radiolabeling (Melby P C et al 1993 JImmunol Methods 159:235-44) or biotinylating (Duplaa C et al 1993 AnalBiochem 229-36) nucleotides, coamplification of a control nucleic acid,and standard curves onto which the experimental results areinterpolated. Quantitation of multiple samples may be speeded up byrunning the assay in an ELISA format where the oligomer of interest ispresented in various dilutions and a spectrophotometric or colorimetricresponse gives rapid quantitation. A definitive diagnosis of this typemay allow health professionals to begin aggressive treatment and preventfurther worsening of the condition. Similarly, further assays can beused to monitor the progress of a patient during treatment. Furthermore,the nucleotide sequences disclosed herein may be used in molecularbiology techniques that have not yet been developed, provided the newtechniques rely on properties of nucleotide sequences that are currentlyknown such as the triplet genetic code, specific base pair interactions,and the like.

Therapeutic Use

Based upon its homology to the PLM-like proteins and its expressionprofile, the polynucleotide encoding HPLP disclosed herein may be usefulin the treatment of diseases associated with aberrant ion transport.

Expression vectors derived from retroviruses, adenovirus, herpes orvaccinia viruses, or from various bacterial plasmids, may be used fordelivery of nucleotide sequences to the targeted organ, tissue or cellpopulation. Methods which are well known to those skilled in the art canbe used to construct recombinant vectors which will express antisense ofthe sequence encoding HPLP. See, for example, the techniques describedin Sambrook et al (supra) and Ausubel et al (supra).

The polynucleotides comprising full length cDNA sequence and/or itsregulatory elements enable researchers to use the sequence encoding HPLPas an investigative tool in sense (Youssoufian H and H F Lodish 1993 MolCell Biol 13:98-104) or antisense (Eguchi et al (1991) Annu Rev Biochem60:631-652) regulation of gene function. Such technology is now wellknown in the art, and sense or antisense oligomers, or larger fragments,can be designed from various locations along the coding or controlregions.

Genes encoding HPLP can be turned off by transfecting a cell or tissuewith expression vectors which express high levels of a desired HPLPfragment. Such constructs can flood cells with untranslatable sense orantisense sequences. Even in the absence of integration into the DNA,such vectors may continue to transcribe RNA molecules until all copiesare disabled by endogenous nucleases. Transient expression may last fora month or more with a non-replicating vector (Mettler I, personalcommunication) and even longer if appropriate replication elements arepart of the vector system.

As mentioned above, modifications of gene expression can be obtained bydesigning antisense molecules, DNA, RNA or PNA, to the control regionsof the sequence encoding HPLP, ie, the promoters, enhancers, andintrons. Oligonucleotides derived from the transcription initiationsite, eg, between -10 and +10 regions of the leader sequence, arepreferred. The antisense molecules may also be designed to blocktranslation of mRNA by preventing the transcript from binding toribosomes. Similarly, inhibition can be achieved using "triple helix"base-pairing methodology. Triple helix pairing compromises the abilityof the double helix to open sufficiently for the binding of polymerases,transcription factors, or regulatory molecules. Recent therapeuticadvances using triplex DNA were reviewed by Gee J E et al (In: Huber B Eand B I Carr (1994) Molecular and Immunologic Approaches, FuturaPublishing Co, Mt Kisco N.Y.).

Ribozymes are enzymatic RNA molecules capable of catalyzing the specificcleavage of RNA. The mechanism of ribozyme action involvessequence-specific hybridization of the ribozyme molecule tocomplementary target RNA, followed by endonucleolytic cleavage. Withinthe scope of the invention are engineered hammerhead motif ribozymemolecules that can specifically and efficiently catalyze endonucleolyticcleavage of the sequence encoding HPLP.

Specific ribozyme cleavage sites within any potential RNA target areinitially identified by scanning the target molecule for ribozymecleavage sites which include the following sequences, GUA, GUU and GUC.Once identified, short RNA sequences of between 15 and 20ribonucleotides corresponding to the region of the target genecontaining the cleavage site may be evaluated for secondary structuralfeatures which may render the oligonucleotide inoperable. Thesuitability of candidate targets may also be evaluated by testingaccessibility to hybridization with complementary oligonucleotides usingribonuclease protection assays.

Antisense molecules and ribozymes of the invention may be prepared byany method known in the art for the synthesis of RNA molecules. Theseinclude techniques for chemically synthesizing oligonucleotides such assolid phase phosphoramidite chemical synthesis. Alternatively, RNAmolecules may be generated by in vitro and in vivo transcription of DNAsequences encoding HPLP. Such DNA sequences may be incorporated into awide variety of vectors with suitable RNA polymerase promoters such asT7 or SP6. Alternatively, antisense cDNA constructs that synthesizeantisense RNA constitutively or inducibly can be introduced into celllines, cells or tissues.

RNA molecules may be modified to increase intracellular stability andhalf-life. Possible modifications include, but are not limited to, theaddition of flanking sequences at the 5' and/or 3' ends of the moleculeor the use of phosphorothioate or 2' O-methyl rather thanphosphodiesterase linkages within the backbone of the molecule. Thisconcept is inherent in the production of PNAs and can be extended in allof these molecules by the inclusion of nontraditional bases such asinosine, queosine and wybutosine as well as acetyl-, methyl-, thio- andsimilarly modified forms of adenine, cytidine, guanine, thymine, anduridine which are not as easily recognized by endogenous endonucleases.

Methods for introducing vectors into cells or tissues include thosemethods discussed infra and which are equally suitable for in vivo, invitro and ex vivo therapy. For ex vivo therapy, vectors are introducedinto stem cells taken from the patient and clonally propagated forautologous transplant back into that same patient as presented in U.S.Pat. Nos. 5,399,493 and 5,437,994, disclosed herein by reference.Delivery by transfection and by liposome are quite well known in theart.

Furthermore, the nucleotide sequences encoding HPLP disclosed herein maybe used in molecular biology techniques that have not yet beendeveloped, provided the new techniques rely on properties of nucleotidesequences that are currently known, including but not limited to suchproperties as the triplet genetic code and specific base pairinteractions.

Detection and Mapping of Related Polynucleotide Sequences

The nucleic acid sequence encoding HPLP can also be used to generatehybridization probes for mapping the naturally occurring genomicsequence. The sequence may be mapped to a particular chromosome or to aspecific region of the chromosome using well known techniques. Theseinclude in situ hybridization to chromosomal spreads, flow-sortedchromosomal preparations, or artificial chromosome constructions such asyeast artificial chromosomes, bacterial artificial chromosomes,bacterial P1 constructions or single chromosome cDNA libraries asreviewed in Price C M (1993; Blood Rev 7:127-34) and Trask B J (1991;Trends Genet 7:149-54).

The technique of fluorescent in situ hybridization of chromosome spreadshas been described, among other places, in Verma et al (1988) HumanChromosomes: A Manual of Basic Techniques, Pergamon Press, New York N.Y.Fluorescent in situ hybridization of chromosomal preparations and otherphysical chromosome mapping techniques may be correlated with additionalgenetic map data. Examples of genetic map data can be found in the 1994Genome Issue of Science (265:1981f). Correlation between the location ofa the sequence encoding HPLP on a physical chromosomal map and aspecific disease (or predisposition to a specific disease) may helpdelimit the region of DNA associated with that genetic disease. Thenucleotide sequences of the subject invention may be used to detectdifferences in gene sequences between normal, carrier or affectedindividuals.

In situ hybridization of chromosomal preparations and physical mappingtechniques such as linkage analysis using established chromosomalmarkers are invaluable in extending genetic maps. A recent example of anSTS based map of the human genome was recently published by theWhitehead-MIT Center for Genomic Research (Hudson T J et al (1995)Science 270:1945-1954). Often the placement of a gene on the chromosomeof another mammalian species such as mouse (Whitehead Institute/MITCenter for Genome Research, Genetic Map of the Mouse, Database Release10, Apr. 28, 1995) may reveal associated markers even if the number orarm of a particular human chromosome is not known. New sequences can beassigned to chromosomal arms, or parts thereof, by physical mapping.This provides valuable information to investigators searching fordisease genes using positional cloning or other gene discoverytechniques. Once a disease or syndrome, such as ataxia telangiectasia(AT), has been crudely localized by genetic linkage to a particulargenomic region, for example, AT to 11q22-23 (Gatti et al (1988) Nature336:577-580), any sequences mapping to that area may representassociated or regulatory genes for further investigation. The nucleotidesequence of the subject invention may also be used to detect differencesin the chromosomal location due to translocation, inversion, etc. amongnormal, carrier or affected individuals.

Pharmaceutical Compositions

The present invention relates to pharmaceutical compositions which maycomprise nucleotides, proteins, antibodies, agonists, antagonists, orinhibitors, alone or in combination with at least one other agent, suchas stabilizing compound, which may be administered in any sterile,biocompatible pharmaceutical carrier, including, but not limited to,saline, buffered saline, dextrose, and water. Any of these molecules canbe administered to a patient alone, or in combination with other agents,drugs or hormones, in pharmaceutical compositions where it is mixed withexcipient(s) or pharmaceutically acceptable carriers. In one embodimentof the present invention, the pharmaceutically acceptable carrier ispharmaceutically inert.

Administration of Pharmaceutical Compositions

Administration of pharmaceutical compositions is accomplished orally orparenterally. Methods of parenteral delivery include topical,intra-arterial (directly to the tumor), intramuscular, subcutaneous,intramedullary, intrathecal, intraventricular, intravenous,intraperitoneal, or intranasal administration. In addition to the activeingredients, these pharmaceutical compositions may contain suitablepharmaceutically acceptable carriers comprising excipients andauxiliaries which facilitate processing of the active compounds intopreparations which can be used pharmaceutically. Further details ontechniques for formulation and administration may be found in the latestedition of "Remington's Pharmaceutical Sciences" (Maack Publishing Co,Easton Pa.).

Pharmaceutical compositions for oral administration can be formulatedusing pharmaceutically acceptable carriers well known in the art indosages suitable for oral administration. Such carriers enable thepharmaceutical compositions to be formulated as tablets, pills, dragees,capsules, liquids, gels, syrups, slurries, suspensions and the like, foringestion by the patient.

Pharmaceutical preparations for oral use can be obtained throughcombination of active compounds with solid excipient, optionallygrinding a resulting mixture, and processing the mixture of granules,after adding suitable auxiliaries, if desired, to obtain tablets ordragee cores. Suitable excipients are carbohydrate or protein fillerssuch as sugars, including lactose, sucrose, mannitol, or sorbitol;starch from corn, wheat, rice, potato, or other plants; cellulose suchas methyl cellulose, hydroxypropylmethyl-cellulose, or sodiumcarboxymethylcellulose; and gums including arabic and tragacanth; andproteins such as gelatin and collagen. If desired, disintegrating orsolubilizing agents may be added, such as the cross-linked polyvinylpyrrolidone, agar, alginic acid, or a salt thereof, such as sodiumalginate.

Dragee cores are provided with suitable coatings such as concentratedsugar solutions, which may also contain gum arabic, talc,polyvinylpyrrolidone, carbopol gel, polyethylene glycol, and/or titaniumdioxide, lacquer solutions, and suitable organic solvents or solventmixtures. Dyestuffs or pigments may be added to the tablets or drageecoatings for product identification or to characterize the quantity ofactive compound, ie, dosage.

Pharmaceutical preparations which can be used orally include push-fitcapsules made of gelatin, as well as soft, sealed capsules made ofgelatin and a coating such as glycerol or sorbitol. Push-fit capsulescan contain active ingredients mixed with a filler or binders such aslactose or starches, lubricants such as talc or magnesium stearate, and,optionally, stabilizers. In soft capsules, the active compounds may bedissolved or suspended in suitable liquids, such as fatty oils, liquidparaffin, or liquid polyethylene glycol with or without stabilizers.

Pharmaceutical formulations for parenteral administration includeaqueous solutions of active compounds. For injection, the pharmaceuticalcompositions of the invention may be formulated in aqueous solutions,preferably in physiologically compatible buffers such as Hanks'solution, Ringer's solution, or physiologically buffered saline. Aqueousinjection suspensions may contain substances which increase theviscosity of the suspension, such as sodium carboxymethyl cellulose,sorbitol, or dextran. Additionally, suspensions of the active compoundsmay be prepared as appropriate oily injection suspensions. Suitablelipophilic solvents or vehicles include fatty oils such as sesame oil,or synthetic fatty acid esters, such as ethyl oleate or triglycerides,or liposomes. Optionally, the suspension may also contain suitablestabilizers or agents which increase the solubility of the compounds toallow for the preparation of highly concentrated solutions.

For topical or nasal administration, penetrants appropriate to theparticular barrier to be permeated are used in the formulation. Suchpenetrants are generally known in the art.

Manufacture and Storage

The pharmaceutical compositions of the present invention may bemanufactured in a manner that is known in the art, eg, by means ofconventional mixing, dissolving, granulating, dragee-making, levigating,emulsifying, encapsulating, entrapping or lyophilizing processes.

The pharmaceutical composition may be provided as a salt and can beformed with many acids, including but not limited to hydrochloric,sulfuric, acetic, lactic, tartaric, malic, succinic, etc. Salts tend tobe more soluble in aqueous or other protonic solvents that are thecorresponding free base forms. In other cases, the preferred preparationmay be a lyophilized powder in 1 mM-50 mM histidine, 0.1%-2% sucrose,2%-7% mannitol at a pH range of 4.5 to 5.5 that is combined with bufferprior to use.

After pharmaceutical compositions comprising a compound of the inventionformulated in a acceptable carrier have been prepared, they can beplaced in an appropriate container and labeled for treatment of anindicated condition. For administration of HPLP, such labeling wouldinclude amount, frequency and method of administration.

Therapeutically Effective Dose

Pharmaceutical compositions suitable for use in the present inventioninclude compositions wherein the active ingredients are contained in aneffective amount to achieve the intended purpose. The determination ofan effective dose is well within the capability of those skilled in theart.

For any compound, the therapeutically effective dose can be estimatedinitially either in cell culture assays, eg, of neoplastic cells, or inanimal models, usually mice, rabbits, dogs, or pigs. The animal model isalso used to achieve a desirable concentration range and route ofadministration. Such information can then be used to determine usefuldoses and routes for administration in humans.

A therapeutically effective dose refers to that amount of protein or itsantibodies, antagonists, or inhibitors which ameliorate the symptoms orcondition. Therapeutic efficacy and toxicity of such compounds can bedetermined by standard pharmaceutical procedures in cell cultures orexperimental animals, eg, ED50 (the dose therapeutically effective in50% of the population) and LD50 (the dose lethal to 50% of thepopulation). The dose ratio of toxic to therapeutic effects is thetherapeutic index, and it can be expressed as the ratio, LD50/ED50.Pharmaceutical compositions which exhibit large therapeutic indices arepreferred. The data obtained from cell culture assays and animal studiesis used in formulating a range of dosage for human use. The dosage ofsuch compounds lies preferably within a range of circulatingconcentrations that include the ED50 with little or no toxicity. Thedosage varies within this range depending upon the dosage form employed,sensitivity of the patient, and the route of administration.

The exact dosage is chosen by the individual physician in view of thepatient to be treated. Dosage and administration are adjusted to providesufficient levels of the active moiety or to maintain the desiredeffect. Additional factors which may be taken into account include theseverity of the disease state, eg, tumor size and location; age, weightand gender of the patient; diet, time and frequency of administration,drug combination(s), reaction sensitivities, and tolerance/response totherapy. Long acting pharmaceutical compositions might be administeredevery 3 to 4 days, every week, or once every two weeks depending onhalf-life and clearance rate of the particular formulation.

Normal dosage amounts may vary from 0.1 to 100,000 micrograms, up to atotal dose of about 1 g, depending upon the route of administration.Guidance as to particular dosages and methods of delivery is provided inthe literature. See U.S. Pat. Nos. 4,657,760; 5,206,344; or 5,225,212.Those skilled in the art will employ different formulations fornucleotides than for proteins or their inhibitors. Similarly, deliveryof polynucleotides or polypeptides will be specific to particular cells,conditions, locations, etc.

The examples below are provided to illustrate the subject invention andare not included for the purpose of limiting the invention.

EXAMPLES

I cDNA Library Construction

The hNT2 cell line exhibits characteristics of a committed neuronalprecursor cell which is at an early stage of development. The hNT2 cellline can be induced by retinoic acid (RA) to differentiate, as describedin Andrews P W (1984) Dev Biol 103:285-293.

For purposes of this invention, hNT2 cells were induced with RA. Themethod used in the present invention involved suspending hNT2 cells inDulbecco's modified Eagle's medium (DMEM) including 10% fetal bovineserum and penicillin/streptomycin, treating the cells with 10 μM RAtwice a week for 5 weeks. The cells were differentially harvested andreplated, and exposed to mitotic inhibitors (1 μM cytosine arabinose, 10μM fluorodeoxyuridine, and 10 μM uridine) for two weeks. The neuronswere again differentially harvested, replated and allowed to maturefurther for 4 weeks in 50% hNT Neuron Conditioned Medium including DMEMand 10% fetal bovine serum. This procedure created cells similar tothose of the postmitotic neuronal cell line of Lee and Pleasure (hNT2-Ncell line) and were named HNT2AGT1 cells.

The mRNA used to prepare the HNT2AGT01 library was isolated byStratagene (La Jolla Calif.) essentially as described below. Firststrand cDNA synthesis was accomplished using an oligo d(T) primer/linkerwhich also contained an XhoI restriction site. Second strand synthesiswas performed using a combination of DNA polymerase I, E. coli ligaseand RNase H, followed by the addition of an EcoRI adaptor to the bluntended cDNA. The EcoRI adapted, double-stranded cDNA was then digestedwith XhoI restriction enzyme and fractionated to obtain sequences whichexceeded 800 bp in size. The cDNAs were inserted into the LAMBDAZAPvector system (Stratagene); then the vector which contains thePBLUESCRIPT phagemid (Stratagene) was transformed into E. coli hostcells strain XL1-BLUEMRF.

The phagemid forms of individual cDNA clones were obtained by the invivo excision process. Enzymes from both PBLUESCRIPT and a cotransformedf1 helper phage nicked the DNA, initiated new DNA synthesis, and createdthe smaller, single-stranded circular phagemid molecules which containedthe cDNA insert. The phagemid DNA was released, purified, and used toreinfect fresh host cells (SOLR, Stratagene). Presence of the phagemidwhich carries the gene for β-lactamase allowed transformed bacteria togrow on medium containing ampicillin.

II Isolation and Sequencing of cDNA Clones

Plasmid DNA was released from the cells and purified using the MiniprepKit (Catalogue # 77468; Advanced Genetic Technologies Corporation,Gaithersburg Md). This kit consists of a 96 well block with reagents for960 purifications. The recommended protocol was employed except for thefollowing changes: 1) the 96 wells were each filled with only 1 ml ofsterile Terrific Broth (Catalog # 22711, LIFE TECHNOLOGIES, GaithersburgMd.) with carbenicillin at 25 mg/L and glycerol at 0.4%; 2) the bacteriawere cultured for 24 hours after the wells were inoculated and thenlysed with 60 μl of lysis buffer; 3) a centrifugation step employing theBeckman GS-6R @2900 rpm for 5 min was performed before the contents ofthe block were added to the primary filter plate; and 4) the optionalstep of adding isopropanol to TRIS buffer was not routinely performed.After the last step in the protocol, samples were transferred to aBeckman 96-well block for storage.

Alternative methods of purifying plasmid DNA include the use of MAGICMINIPREPS DNA Purification System (Catalogue #A7100, Promega, MadisonWis.) or QIAWELL™-8 Plasmid, QIAWELL PLUS DNA and QIAWELL ULTRA DNAPurification Systems (QIAGEN® Chatsworth Calif.).

The cDNAs were sequenced by the method of Sanger F and A R Coulson(1975; J Mol Biol 94:441f), using a Hamilton Micro Lab 2200 (Hamilton,Reno Nev.) in combination with four Peltier Thermal Cyclers (PTC200 fromMJ Research, Watertown Ma.) and Applied Biosystems 377 or 373 DNASequencing Systems (Perkin Elmer) and reading frame was determined.

III Homology Searching of cDNA Clones and Their Deduced Proteins

Each cDNA was compared to sequences in GenBank using a search algorithmdeveloped by Applied Biosystems and incorporated into the INHERIT™ 670Sequence Analysis System. In this algorithm, Pattern SpecificationLanguage (TRW Inc, Los Angeles Calif.) was used to determine regions ofhomology. The three parameters that determine how the sequencecomparisons run were window size, window offset, and error tolerance.Using a combination of these three parameters, the DNA database wassearched for sequences containing regions of homology to the querysequence, and the appropriate sequences were scored with an initialvalue. Subsequently, these homologous regions were examined using dotmatrix homology plots to distinguish regions of homology from chancematches. Smith-Waterman alignments were used to display the results ofthe homology search.

Peptide and protein sequence homologies were ascertained using theINHERIT 670 Sequence Analysis System in a way similar to that used inDNA sequence homologies. Pattern Specification Language and parameterwindows were used to search protein databases for sequences containingregions of homology which were scored with an initial value. Dot-matrixhomology plots were examined to distinguish regions of significanthomology from chance matches.

BLAST, which stands for Basic Local Alignment Search Tool (Altschul S F(1993) J Mol Evol 36:290-300; Altschul, S F et al (1990) J Mol Biol215:403-10), was used to search for local sequence alignments. BLASTproduces alignments of both nucleotide and amino acid sequences todetermine sequence similarity. Because of the local nature of thealignments, BLAST is especially useful in determining exact matches orin identifying homologs. BLAST is useful for matches which do notcontain gaps. The fundamental unit of BLAST algorithm output is theHigh-scoring Segment Pair (HSP).

An HSP consists of two sequence fragments of arbitrary but equal lengthswhose alignment is locally maximal and for which the alignment scoremeets or exceeds a threshold or cutoff score set by the user. The BLASTapproach is to look for HSPs between a query sequence and a databasesequence, to evaluate the statistical significance of any matches found,and to report only those matches which satisfy the user-selectedthreshold of significance. The parameter E establishes the statisticallysignificant threshold for reporting database sequence matches. E isinterpreted as the upper bound of the expected frequency of chanceoccurrence of an HSP (or set of HSPs) within the context of the entiredatabase search. Any database sequence whose match satisfies E isreported in the program output.

IV Northern Analysis

Northern analysis is a laboratory technique used to detect the presenceof a transcript of a gene and involves the hybridization of a labelednucleotide sequence to a membrane on which RNAs from a particular celltype or tissue have been bound (Sambrook et al. supra).

Analogous computer techniques using BLAST (Altschul S F 1993 and 1990,supra) are used to search for identical or related molecules innucleotide databases such as GenBank or the LIFESEQ database (Incyte,Palo Alto Calif.). This analysis is much faster than multiple,membrane-based hybridizations. In addition, the sensitivity of thecomputer search can be modified to determine whether any particularmatch is categorized as exact or homologous.

The basis of the search is the product score which is defined as:

    % sequence identity×% maximum BLAST score/100

and it takes into account both the degree of similarity between twosequences and the length of the sequence match. For example, with aproduct score of 40, the match will be exact within a 1-2% error; and at70, the match will be exact. Homologous molecules are usually identifiedby selecting those which show product scores between 15 and 40, althoughlower scores may identify related molecules.

The results of the search are reported as a list of libraries in whichthe HPLP encoding sequence occurs. Abundance and percentage abundance ofthe HPLP encoding sequence are also reported. Abundance directlyreflects the number of times a particular transcript is represented in acDNA library, and percent abundance is abundance divided by the totalnumber of sequences examined in the cDNA library.

V Extension of the Sequence Encoding HPLP

The nucleic acid sequence of SEQ ID NO:2 is used to designoligonucleotide primers for extending a partial nucleotide sequence tofull length or for obtaining 5' sequence from genomic libraries. Oneprimer is synthesized to initiate extension in the antisense direction(XLR) and the other is synthesized to extend sequence in the sensedirection (XLF). Primers allow the extension of the known sequence"outward" generating amplicons containing new, unknown nucleotidesequence for the region of interest The initial primers are designedfrom the cDNA using OLIGO 4.06 Primer Analysis Software (NationalBiosciences), or another appropriate program, to be 22-30 nucleotides inlength, to have a GC content of 50% or more, and to anneal to the targetsequence at temperatures about 68°-72° C. Any stretch of nucleotideswhich would result in hairpin structures and primer-primer dimerizationsis avoided.

The original, selected cDNA libraries, or a human genomic library areused to extend the sequence; the latter is most useful to obtain 5'upstream regions. If more extension is necessary or desired, additionalsets of primers are designed to further extend the known region.

By following the instructions for the XL-PCR kit (Perkin Elmer) andthoroughly mixing the enzyme and reaction mix, high fidelityamplification is obtained. Beginning with 40 pmol of each primer and therecommended concentrations of all other components of the kit, PCR isperformed using the Peltier Thermal Cycler (PTC200; MJ Research,Watertown Ma.) and the following parameters:

    ______________________________________    Step 1       94° C. for 1 min (initial denaturation)    Step 2       65° C. for 1 min    Step 3       68° C. for 6 min    Step 4       94° C. for 15 sec    Step 5       65° C. for 1 min    Step 6       68° C. for 7 min    Step 7       Repeat step 4-6 for 15 additional cycles    Step 8       94° C. for 15 sec    Step 9       65° C. for 1 min    Step 10      68° C. for 7:15 min    Step 11      Repeat step 8-10 for 12 cycles    Step 12      72° C. for 8 min    Step 13      4° C. (and holding)    ______________________________________

A 5-10 μl aliquot of the reaction mixture is analyzed by electrophoresison a low concentration (about 0.6-0.8%) agarose mini-gel to determinewhich reactions were successful in extending the sequence. Bands thoughtto contain the largest products were selected and cut out of the gel.Further purification involves using a commercial gel extraction methodsuch as QIAQUICK (QIAGEN Inc). After recovery of the DNA, Klenow enzymewas used to trim single-stranded, nucleotide overhangs creating bluntends which facilitate religation and cloning.

After ethanol precipitation, the products are redissolved in 13 μl ofligation buffer, 1 μl T4-DNA ligase (15 units) and 1 μl T4polynucleotide kinase are added, and the mixture is incubated at roomtemperature for 2-3 hours or overnight at 16° C. Competent E. coli cells(in 40 μl of appropriate media) are transformed with 3 μl of ligationmixture and cultured in 80 μl of SOC medium (Sambrook J et al, supra).After incubation for one hour at 37° C., the whole transformationmixture is plated on Luria Bertani (LB)-agar (Sambrook J et al, supra)containing 2×Carb. The following day, several colonies are randomlypicked from each plate and cultured in 150 μl of liquid LB/2×Carb mediumplaced in an individual well of an appropriate, commercially-available,sterile 96-well microtiter plate. The following day, 5 μl of eachovernight culture is transferred into a non-sterile 96-well plate andafter dilution 1:10 with water, 5 μl of each sample is transferred intoa PCR array.

For PCR amplification, 18 μl of concentrated PCR reaction mix (3.3×)containing 4 units of rTth DNA polymerase, a vector primer and one orboth of the gene specific primers used for the extension reaction areadded to each well. Amplification is performed using the followingconditions:

    ______________________________________    Step 1     94° C. for 60 sec    Step 2     94° C. for 20 sec    Step 3     55° C. for 30 sec    Step 4     72° C. for 90 sec    Step 5     Repeat steps 2-4 for an additional 29 cycles    Step 6     72° C. for 180 sec    Step 7     4° C. (and holding)    ______________________________________

Aliquots of the PCR reactions are run on agarose gels together withmolecular weight markers. The sizes of the PCR products are compared tothe original partial cDNAs, and appropriate clones are selected, ligatedinto plasmid and sequenced.

VI Labeling and Use of Hybridization Probes

Hybridization probes derived from SEQ ID NO:2 are employed to screencDNAs, genomic DNAs or mRNAs. Although the labeling of oligonucleotides,consisting of about 20 base-pairs, is specifically described,essentially the same procedure is used with larger cDNA fragments.Oligonucleotides are designed using state-of-the-art software such asOLIGO 4.06 (National Biosciences), labeled by combining 50 pmol of eacholigomer and 250 mCi of γ-³² P! adenosine triphosphate (Amersham,Chicago Ill.) and T4 polynucleotide kinase (DuPont NEN Boston Mass.).The labeled oligonucleotides are substantially purified with SEPHADEXG-25 super fine resin column (Pharmacia). A portion containing 10⁷counts per minute of each of the sense and antisense oligonucleotides isused in a typical membrane based hybridization analysis of human genomicDNA digested with one of the following endonucleases (Ase I, Bgl II, EcoRI, Pst I, Xba 1, or Pvu II; DuPont NEN).

The DNA from each digest is fractionated on a 0.7 percent agarose geland transferred to nylon membranes (Nytran Plus, Schleicher & Schuell,Durham N.H.). Hybridization is carried out for 16 hours at 40° C. Toremove nonspecific signals, blots are sequentially washed at roomtemperature under increasingly stringent conditions up to 0.1×salinesodium citrate and 0.5% sodium dodecyl sulfate. After XOMAT AR film(Kodak, Rochester N.Y.) is exposed to the blots in a Phosphoimagercassette (Molecular Dynamics, Sunnyvale Calif.) for several hours,hybridization patterns are compared visually.

VII Antisense Molecules

The sequence encoding HPLP, or any part thereof, is used to inhibit invivo or in vitro expression of naturally occurring sequence. Althoughuse of antisense oligonucleotides, comprising about 20 base-pairs, isspecifically described, essentially the same procedure is used withlarger cDNA fragments. An oligonucleotide complementary to a portion ofthe coding sequence of HPLP as shown in SEQ ID NO:2 is used to inhibitexpression of naturally occurring sequence. The complementaryoligonucleotide is designed from the most unique 5' sequence and usedeither to inhibit transcription by preventing promoter binding to theupstream nontranslated sequence or translation of a transcript encodingHPLP by preventing the ribosome from binding. Using an appropriateportion of the leader and 5' sequence of SEQ ID NO:2, an effectiveantisense oligonucleotide includes any 15-20 nucleotides spanning theregion which translates into the signal or early coding sequence of thepolypeptide as shown in FIG. 1.

VIII Expression of HPLP

Expression of HPLP is accomplished by subcloning the cDNAs intoappropriate vectors and transfecting the vectors into host cells. Inthis case, the cloning vector, PSPORT, previously used for thegeneration of the cDNA library is used to express HPLP in E. coli.Upstream of the cloning site, this vector contains a promoter forβ-galactosidase, followed by sequence containing the amino-terminal Metand the subsequent 7 residues of β-galactosidase. Immediately followingthese eight residues is a bacteriophage promoter useful fortranscription and a linker containing a number of unique restrictionsites.

Induction of an isolated, transfected bacterial strain with IPTG usingstandard methods produces a fusion protein which consists of the firstseven residues of β-galactosidase, about 5 to 15 residues of linker, andthe full length HPLP. The signal sequence directs the secretion of HPLPinto the bacterial growth media which can be used directly in thefollowing assay for activity.

IX Assay for HPLP Activity

The channel-forming ability of HPLP is assayed by monitoring efflux ofCl⁻ or K⁺ ions from vesicles containing HPLP subjected to atransmembrane ion potential. HPLP and mitochondrial cytochrome Coxidase, a proton pump, are reconstituted into lipid vesicles bysonication. ³⁶ Cl⁻ or ⁴² K⁺ is then incorporated into the vesicles bypassive diffusion, incubating the vesicles in a solution containing ³⁶Cl!-potassium chloride or ⁴² K!-potassium chloride for several hours.The vesicles are then dispersed in an appropriate reaction buffer.Addition of ascorbate and cytochrome C initiates proton uptake into thevesicles generating an interior-positive membrane potential. The voltagegenerated across the membrane activates gating of the HPLP ion channel.At predetermined times, aliquots of the vesicle-containing solution areremoved from the reaction buffer and filtered through 0.2 μmembranefilters (Millipore, Marlboro Mass.). The vesicles are retained on thefilters. The filters are rinsed and dried. Radioactivity on the filtersis measured in a scintillation counter. The decrease in radioactivity onthe filters as a function of reaction time gives a measure of the rateof Cl⁻ or K⁺ efflux through the voltage-activated HPLP ion channel.

X Production of HPLP Specific Antibodies

HPLP substantially purified using PAGE electrophoresis (Sambrook,supra,) is used to immunize rabbits and to produce antibodies usingstandard protocols. The amino acid sequence translated from HPLP isanalyzed using DNAStar software (DNAStar Inc) to determine regions ofhigh immunogenicity and a corresponding oligopeptide is synthesized andused to raise antibodies by means known to those of skill in the art.Analysis to select appropriate epitopes, such as those near theC-terminus or in hydrophilic regions (shown in FIG. 3) is described byAusubel F M et al (supra).

Typically, the oligopeptides are 15 residues in length, synthesizedusing an Applied Biosystems Peptide Synthesizer Model 431A usingfmoc-chemistry, and coupled to keyhole limpet hemocyanin (KLH, Sigma) byreaction with M-maleimidobenzoyl-N-hydroxysuccinimide ester (MBS;Ausubel F M et al, supra). Rabbits are immunized with theoligopeptide-KLH complex in complete Freund's adjuvant. The resultingantisera are tested for antipeptide activity, for example, by bindingthe peptide to plastic, blocking with 1% BSA, reacting with rabbitantisera, washing, and reacting with radioiodinated, goat anti-rabbitIgG.

XI Purification of Naturally Occurring HPLP Using Specific Antibodies

Naturally occurring or recombinant HPLP is substantially purified byimmunoaffinity chromatography using antibodies specific for HPLP. Animmunoaffinity column is constructed by covalently coupling HPLPantibody to an activated chromatographic resin such as CnBr-activatedSEPHAROSE (Pharmacia Biotech). After the coupling, the resin is blockedand washed according to the manufacturer's instructions.

Membrane fractions from cells expressing HPLP are prepared by methodswell known in the art. Alternatively, a recombinant HPLP fragmentcontaining an appropriate signal sequence may be secreted in usefulquantity into the medium in which transfected cells are grown.

The HPLP-containing preparation is passed over the immunoaffinitycolumn, and the column is washed under conditions that allow thepreferential absorbance of HPLP (eg, high ionic strength buffers in thepresence of detergent). The column is eluted under conditions thatdisrupt antibody/HPLP binding (eg, a buffer of pH 2-3 or a highconcentration of a chaotrope such as urea or thiocyanate ion), and HPLPis collected.

All publications and patents mentioned in the above specification areherein incorporated by reference. Various modifications and variationsof the described method and system of the invention will be apparent tothose skilled in the art without departing from the scope and spirit ofthe invention. Although the invention has been described in connectionwith specific preferred embodiments, it should be understood that theinvention as claimed should not be unduly limited to such specificembodiments. Indeed, various modifications of the described modes forcarrying out the invention which are obvious to those skilled inmolecular biology or related fields are intended to be within the scopeof the following claims.

    __________________________________________________________________________    #             SEQUENCE LISTING    - (1) GENERAL INFORMATION:    -    (iii) NUMBER OF SEQUENCES: 6    - (2) INFORMATION FOR SEQ ID NO:1:    -      (i) SEQUENCE CHARACTERISTICS:    #acids    (A) LENGTH: 95 amino              (B) TYPE: amino acid              (C) STRANDEDNESS: single              (D) TOPOLOGY: linear    -     (ii) MOLECULE TYPE: peptide    -    (vii) IMMEDIATE SOURCE:              (A) LIBRARY:              (B) CLONE: Consensus    -            (xi) SEQUENCE DESCRIPT - #ION: SEQ ID NO:1:    #Leu Leu Ala Pro Met Valal Phe Leu Cys Ser    #                 15    #Met Asp Pro Phe His Tyrlu Lys Glu Lys Glu    #             30    #Val Phe Ala Val Val Leurg Ile Gly Gly Leu    #         45    #Arg Arg Cys Lys Cys Sereu Leu Ile Leu Ser    #     60    #Glu Glu Ala Gln Val Glurg Ala Pro Gly Asp    # 80    #Gln Lys Ala Glu Asnla Asn Ala Thr Glu Pro    #                 95    - (2) INFORMATION FOR SEQ ID NO:2:    -      (i) SEQUENCE CHARACTERISTICS:    #pairs    (A) LENGTH: 591 base              (B) TYPE: nucleic acid              (C) STRANDEDNESS: single              (D) TOPOLOGY: linear    -     (ii) MOLECULE TYPE: cDNA    -    (vii) IMMEDIATE SOURCE:              (A) LIBRARY:              (B) CLONE: Consensus    -            (xi) SEQUENCE DESCRIPT - #ION: SEQ ID NO:2:    #GCAGATCTAT    60CAGAGGT GGATGGGGCT TGAAAAGGGG GTTCAAGGCA    #CCCCCATGGT   120ATGGAGT TGGTGCTGGT CTTCCTCTGC AGCCTGCTGG    #ATTACCAGAC   180GCTGAAA AGGAGAAGGA AATGGACCCT TTTCATTATG    #TCCTCCTTAT   240GGACTGG TGTTCGCTGT GGTCCTCTTC TCGGTTGGGA    #GAGATGAGGA   300TGCAAGT GCAGTTTCAA TCAGAAGCCC CGGGCCCCAG    #CAGAGAACTG   360AACCTCA TCACCGCCAA TGCAACAGAG CCCCAGAAAG    #CCTTTGGATG   420AGGTGGA AGCCTCTGGA ACCTGAGGCG GCTGCTTGAA    #CAGGAGAAGC   480TTAAGAA AACCGGCCAC TTCAGCAACA GCCCTTTCCC    #CTGATGATGC   540GTCCCCC ACCCTATCCC CTCTAACACC ATTCCTCCAC    #T            591CTCCCCA CTGCAGCCTG CGGTCCTGCC CACCTCCCGA    - (2) INFORMATION FOR SEQ ID NO:3:    -      (i) SEQUENCE CHARACTERISTICS:    #acids    (A) LENGTH: 92 amino              (B) TYPE: amino acid              (C) STRANDEDNESS: single              (D) TOPOLOGY: linear    -     (ii) MOLECULE TYPE: peptide    -    (vii) IMMEDIATE SOURCE:              (A) LIBRARY: GenBank              (B) CLONE: 108084    -            (xi) SEQUENCE DESCRIPT - #ION: SEQ ID NO:3:    #Cys Val Gly Phe Leu Thris Ile Leu Val Leu    #                 15    #Asp Pro Phe Thr Tyr Aspla Pro Gln Glu His    #             30    #Ile Ala Gly Ile Leu Phele Gly Gly Leu Ile    #         45    #Arg Cys Arg Cys Lys Phele Val Leu Ser Arg    #     60    #Glu Glu Glu Gly Thr Phehr Gly Glu Pro Asp    # 80    #Arg Arger Ser Ile Arg Arg Leu Ser Thr Arg    #                 90    - (2) INFORMATION FOR SEQ ID NO:4:    -      (i) SEQUENCE CHARACTERISTICS:    #acids    (A) LENGTH: 87 amino              (B) TYPE: amino acid              (C) STRANDEDNESS: single              (D) TOPOLOGY: linear    -     (ii) MOLECULE TYPE: peptide    -    (vii) IMMEDIATE SOURCE:              (A) LIBRARY: GenBank              (B) CLONE: 1085026    -            (xi) SEQUENCE DESCRIPT - #ION: SEQ ID NO:4:    #Phe Leu Ala Gly Phe Proeu Gly Leu Leu Val    #                 15    #Asn Ser Pro Phe Tyr Tyrsp Leu Glu Asp Lys    #             30    #Ile Cys Ala Gly Val Leuln Val Gly Gly Leu    #         45    #Ala Lys Cys Lys Cys Lysle Ile Val Met Ser    #     60    #Glu Thr Pro Pro Leu Ilely His His Pro Gly    # 80    -  Thr Pro Gly Ser Ala Gln Ser                     85    - (2) INFORMATION FOR SEQ ID NO:5:    -      (i) SEQUENCE CHARACTERISTICS:    #acids    (A) LENGTH: 87 amino              (B) TYPE: amino acid              (C) STRANDEDNESS: single              (D) TOPOLOGY: linear    -     (ii) MOLECULE TYPE: peptide    -    (vii) IMMEDIATE SOURCE:              (A) LIBRARY: GenBank              (B) CLONE: 951423    -            (xi) SEQUENCE DESCRIPT - #ION: SEQ ID NO:5:    #Val Leu Ala Gly Leu Proys Ala Phe Leu Leu    #                 15    #Gly Ser Pro Phe Tyr Tyrly Pro Val Asp Lys    #             30    #Ile Phe Gly Gly Leu Leuln Leu Gly Gly Met    #         45    #Gly Lys Cys Lys Cys Argla Met Ala Leu Ser    #     60    #Lys Val Thr Pro Leu Ileer Ser Leu Pro Glu    # 80    -  Thr Pro Gly Ser Ala Ser Thr                     85    - (2) INFORMATION FOR SEQ ID NO:6:    -      (i) SEQUENCE CHARACTERISTICS:    #acids    (A) LENGTH: 58 amino              (B) TYPE: amino acid              (C) STRANDEDNESS: single              (D) TOPOLOGY: linear    -     (ii) MOLECULE TYPE: peptide    -    (vii) IMMEDIATE SOURCE:              (A) LIBRARY: GenBank              (B) CLONE: 51112    -            (xi) SEQUENCE DESCRIPT - #ION: SEQ ID NO:6:    #Phe Glu Tyr Asp Tyr Gluly Thr Glu Asn Pro    #                 15    #Gly Leu Ala Phe Val Vally Leu Ile Phe Ala    #             30    #Arg Cys Gly Gly Gly Lyseu Ser Lys Arg Phe    #         45    -  Lys His Arg Gln Val Asn Glu Asp Glu Leu    #     55    __________________________________________________________________________

We claim:
 1. A hybridization probe comprising SEQ ID NO:2.
 2. A methodfor the detection of polynucleotides encoding SEQ ID NO:1 in abiological sample comprising the steps of:a) hybridizing the probe ofclaim 1 to nucleic acid material, thereby forming a hybridizationcomplex, and b) detecting said hybridization complex, wherein thepresence of said complex correlates with the presence of apolynucleotide encoding human phospholemman-like protein in saidbiological sample.
 3. The method of claim 2 wherein beforehybridization, the nucleic acid material of the biological sample isamplified by the polymerase chain reaction.